Measurement of microbial biomass or other soil analytes is difficult because of the large amount of particulate matter that is irrelevant to the measurement of these analytes and because the color of an extract may preclude assaying for analytes by methods such as spectrophotometery, turbidity, nephalometry and visual comparison. One of the most difficult parameters to measure is Microbial Biomass which is an excellent indicator of soil and compost quality and is a predictor of soil fertility. Soil microbes recycle the organic matter in soil and convert it into forms that can be utilized by plants. Bacteria represent the most numerous of the microbial life in soil and serve as the bottom rung of the microbial soil food chain which consists of bacteria, fungi, protozoa, algae and nematodes. Abundant microbial life indicates that the nutrient levels of soil are sufficient and balanced and that there is an absence of significant levels of deleterious or poisonous substances such as heavy metals or high concentrations of salts.
Studies have revealed that microbial biomass is a predictor of soil fecundity and correlates highly with other predictors of fecundity such as organic carbon and soluble organic carbon and crop yield. However, tests and test standardization to establish the microbial content of soils are not extensively utilized in large part due to the fact that existing methods are laboratory tests and carry a high cost or have poor performance.
Currently, several laboratories provide commercial in-house services to estimate the numbers of various different types of microbes in soil. These estimates are based upon laboratory tests that are costly, labor intensive and results are not available for 7-21 days. Because less than 10% of soil microbes can be cultured and then only with great difficulty and time, analyses may be performed by direct counting using a microscope and a diluted sample on a slide. The slide can be difficult to read because microbes are attached to the soil particles, and expertise is required to distinguish between bacteria, fungi and protozoa, rendering these tests prohibitively expensive for use as routine quality control. In addition, soil samples must be transported to the laboratory for analysis during which time it has been shown that microbial biomass can rapidly decline, and it can take days or weeks for the results to be reported. These methods are not practical for estimation of the microbial content of composts and compost extracts, which must be used within one or two days of formulation. Further, the results are not consistent from lab to lab, due to the subjective nature of visual counts. Analytical methods of microbial biomass, such as phospholipid fatty acid analysis (PLFA) and carbon fumigation cost upwards of $80: PLFA calculates the weight of the various phospholipids of microbes which is very useful in indicating the microbial composition as different phospholipids are associated with certain species. Microbial biomass can be back calculated from the total weight of phospholipids using a factor of about 100. This method has been shown to have about a 70% correlation with the carbon fumigation method, which is the gold standard for estimation of microbial biomass. The carbon fumigation method costs about $500 and is provided by only a few U.S. labs. It measures the amount of carbon dioxide produced by actively metabolizing microbes over a set period of time, usually a week: This test is done is quadruplicate as there is enough variation in one sample that only an average is considered accurate. Microbial biomass is back calculated from this using a factor. Both of these methods do not measure biomass but infer it from microbial parameters, thus there is not excellent correlation from method to method. For the microbial count, PLFA and carbon fumigation methods there is so much lab to lab variation that one cannot compare results from one lab to those of another, although each lab's method appears rather reproducible. The lab to lab variability of these methods is due to the fact that they are complex procedures performed slightly differently from lab to lab, they are technique dependent and the microbial biomass measured is undoubted heavily influenced by the various preparation procedures that are used. These procedures affect the microbes which are living creatures. The carbon burst method measures CO2 generation by microbial metabolism over a 24 hour or longer period. It utilizes a dried sample. It is well known that drying kills metabolically active and dividing microbes which is the case during periods of plant growth. Thus it measures the microbial activity of the least metabolically active microbes. It can also be used to measure anaerobic respiration in a tight container.
Bacteria are typically the most abundant and diverse microbial component of soil. A standard laboratory technique for quantitation of bacteria is based upon spectrophotometric measurement of turbidity within a solution. However, this method is problematic for measurement of the bacterial content of soil samples because the particles and pigments in soil also contribute to turbidity, reflectance and/or transmittance measurements. In addition, many microbes in soil are firmly attached to the soil particles and do not readily go into solution. Further, it is not practical to apply these methods to a field test to assess microbial biomass in soil because of the need for a turbidometer or spectrophotometer, which precludes efficient use in the field. In order to be of most use to agriculturists, low cost accurate estimates of microbial numbers are required on-site and within minutes to hours of sampling, e.g. to determine whether a new treatment increased microbial growth, how much to dilute a compost extract, or whether further fertilizer treatments are necessary.
The device described in U.S. Pat. No. 9,315,849, is useful in a method for separating bacteria from soil particles and estimating bacterial biomass by turbidity. In contrast, it would be preferable to have a method which optimizes the extraction procedure to allow the extraction of both bacteria and protozoa. The extracted microbes are colored. The intensity of the color correlates with the microbial biomass, allowing microbial biomass to be estimated from the intensity of the color, estimated from spectrophotometry or by visual methods.
There remains a need for easy, accurate, and fast estimation of soil microbial load that does not require laboratory instrumentation and can be practiced in a field setting, because microbial biomass is the single best predictor of soil quality.
Therefore, it is the object of the present invention to provide a method and devices for easy, accurate, and fast estimation of soil microbial load.
It is another object of the present invention to provide methods for easy, accurate, and fast estimation of soil microbial load.
It is yet another object of the present invention to provide kits for easy, accurate, and fast estimation of soil microbial load.